Abstract

Colorectal cancer is still one of the deadliest diseases in western countries, particularly for those patients in which the diagnosis is performed at advanced stage. To identify useful predictive biomarkers of the outcome, we assessed using immunohistochemistry the expression of class III (TUBB3) and class V β-tubulin (TUBB6) in a retrospective clinical study on 180 colorectal cancer patients. When stratified by gender, we noticed that only in females a significant link between poor survival, metastatic disease and the expression of both TUBB3 and TUBB6 was found, while in males no relationship was detectable. In order to get insights into this phenomenon, we analyzed the expression levels of both tubulin isotypes in a panel of 22 colorectal cancer cell lines (13 from males and 9 from females) in basal conditions and after serum starvation, as a stimulus capable to activate the expression of this survival pathway. Male cell lines exhibited a relative resistance to serum starvation and higher levels of the tubulin isotypes, while only in female there was the activation of the pathway under stressing conditions. These findings indicate that in males the survival pathway related to TUBB3/TUBB6 is constitutively active, while in females it is activated by environmental stressors. In order to test if androgens could be responsible for the constitutive activation of this pathway in males, we analyzed the expression of androgen receptor (AR) in the 22 cancer cell lines. A statistically significant correlation was found between AR and TUBB3 (R= 0. 53, p= 0.0062) and TUBB6 (R=0.41; p=0.048) at the gene level. The pivotal role of androgen receptor in driving this pathway was demonstrated after constitutive silencing of AR in two models (SW480 and COLO-320) with high AR and TUBB3/TUBB6 expression. Knock-down of AR was associated to a significant reduction of the expression of both TUBB3 and TUBB6, along with decreased clonogenic activity and increased sensitivity to agents such as oxaliplatin and SN-38. Mortality for colorectal cancer is different by gender and higher in males. The molecular basis for such difference is unknown. In this study, we have demonstrated that in female cell lines (and probably patients) the activation of the survival pathway mediated by TUBB3 and TUBB6 is driven by microenvironmental stress like serum starvation. Those tumors which grow in the most compelling microenvironments will overexpress both proteins and will exhibit an aggressive behavior. In male this correlation is lost since the survival pathway is constitutively active and not dependent on microenvironmental conditioning. This makes that the disease intrinsically more aggressive and less sensitive to treatments. The involvement of AR in the activation of this survival pathway opens the avenue to clinical trials assessing the efficacy of antiandrogens in the management of advanced colorectal cancer.